β3-Adrenoceptor Control the Cystic Fibrosis Transmembrane Conductance Regulator through a cAMP/Protein Kinase A-independent Pathway

Abstract

In human cardiac myocytes, we have previously identified a functional ␤ 3 -adrenoceptor in which stimulation reduces action potential duration. Surprisingly, in cardiac biopsies obtained from cystic fibrosis patients, ␤ 3 -adrenoceptor agonists produced no effects on action potential duration. This result suggests the involvement of cystic fibrosis transmembrane conductance regulator (CFTR) chloride current in the electrophysiological effects of ␤ 3 -adrenoceptor stimulation in non-cystic fibrosis tissues. We therefore investigated the control of CFTR activity by human ␤ 3 -adrenoceptors in a recombinant system: A549 human cells were intranuclearly injected with plasmids encoding CFTR and ␤ 3 -adrenoceptors. CFTR activity was functionally assayed using the 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescent probe and the patch-clamp technique. Injection of CFTR-cDNA alone led to the expression of a functional CFTR protein activated by cAMP or cGMP. Co-expression of CFTR (but not of mutated ⌬F508-CFTR) with high levels of ␤ 3 -adrenoceptor produced an increased halide permeability under base-line conditions that was not further sensitive to cAMP or ␤ 3 -adrenoceptor stimulation. Patchclamp experiments confirmed that CFTR channels were permanently activated in cells co-expressing CFTR and a high level of ␤ 3 -adrenoceptor. Permanent CFTR activation was not associated with elevated intracellular cAMP or cGMP levels. When the expression level of ␤ 3 -adrenoceptor was lowered, CFTR was not activated under base-line conditions but became sensitive to ␤ 3 -adrenoceptor stimulation (isoproterenol plus nadolol, SR 58611, or CGP 12177). This later effect was not prevented by protein kinase A inhibitors. Our results provide molecular evidence that CFTR but not mutated ⌬F508-CFTR is regulated by ␤ 3 -adrenoceptors expression through a protein kinase A-independent pathway.

Transcriptional Adaptation to Cystic Fibrosis Transmembrane Conductance Regulator Deficiency

“…In contrast to the transport/ receptors that were induced, ADRB3, a G protein-coupled transmembrane protein mediating cAMP production was decreased in CFTR(Ϫ) mice. ADRB3 is co-expressed with CFTR in airway epithelium (45) and may be functionally coupled to CFTR via cAMP-independent pathways (46). ␤2-Adrenergic receptors directly interact with CFTR via the Na(ϩ)/H(ϩ) exchanger regulatory factor to form a signal transduction complex (47).…”

Discovery of α-Aminoazaheterocycle-Methylglyoxal Adducts as a New Class of High-Affinity Inhibitors of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels

“…The slope of the straight line reflecting the membrane permeability to halides (noted as p in Fig. 7 and measured per minute) was used as an index of CFTR activity (Leblais et al, 1999).…”

β3-adrenergic receptor activation increases human atrial tissue contractility and stimulates the L-type Ca2+ current

“…However, in rat and mice adipocytes, in addition to their coupling to G s , β 3 -ARs couple to G i/o proteins and inhibit adenylyl cyclase (60,61). In the human A549 lung epithelial cell line and in the human colonic carcinoma cell line T84, β 3 -ARs appear to be exclusively linked to G i/o proteins (18,62). Functional response to β 3 -AR…”

“…Because contractility of a trabeculum is a more integrated response than I Ca,L amplitude in a single myocyte, many other parameters will participate in the inotropic response to β 3 -AR agonists. One of these is the action potential duration, which is affected not only by β 3 -AR regulation of I Ca,L but also by the effect of the receptor on other ion channels, such as I Ks (51), the rapid component of the delayed rectifier potassium current (I Kr ) (52), the hyperpolarization-activated pacemaker current I f (54), and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl -current (18). Other downstream factors include intracellular Ca 2+ transient and sarcoplasmic reticulum function and the phosphorylation status of key proteins such as phospholamban, ryanodine receptor, and contractile proteins.…”

“…However, β 3 -AR agonists were shown to inhibit contractility in human endomyocardial biopsies from transplanted hearts (8,16) and in left ventricular samples from failing and nonfailing explanted hearts (11), an effect mediated by G i/o proteins and NO production via activation of endothelial type-3 NO synthase (8,16,17). This effect was accompanied by a shortening of the action potential (8,18). A similar finding was obtained with BRL37344, a β 3 -AR agonist, in Langendorff-perfused isolated guinea pig hearts (19) and was accompanied by a decrease in Ca 2+ transients.…”